ABSTRACT

The iron-directed, coordinate regulation of genes depends on the fur (ferric uptake regulator) gene product, which acts as an iron-responsive, transcriptional repressor protein. To investigate the biological function of a fur homolog in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1, a fur knockout strain (FUR1) was generated by suicide plasmid integration into this gene and characterized using phenotype assays, DNA microarrays containing 691 arrayed genes, and two-dimensional polyacrylamide gel electrophoresis. Physiological studies indicated that FUR1 was similar to the wild-type strain when they were compared for anaerobic growth and reduction of various electron acceptors. Transcription profiling, however, revealed that genes with predicted functions in electron transport, energy metabolism, transcriptional regulation, and oxidative stress protection were either repressed (ccoNQ, etrA, cytochrome b and c maturation-encoding genes, qor, yiaY, sodB, rpoH, phoB, and chvI) or induced (yggW, pdhC, prpC, aceE, fdhD, and ppc) in the fur mutant. Disruption of fur also resulted in derepression of genes (hxuC, alcC, fhuA, hemR, irgA, and ompW) putatively involved in iron uptake. This agreed with the finding that the fur mutant produced threefold-higher levels of siderophore than the wild-type strain under conditions of sufficient iron. Analysis of a subset of the FUR1 proteome (i.e., primarily soluble cytoplasmic and periplasmic proteins) indicated that 11 major protein species reproducibly showed significant (P < 0.05) differences in abundance relative to the wild type. Protein identification using mass spectrometry indicated that the expression of two of these proteins (SodB and AlcC) correlated with the microarray data. These results suggest a possible regulatory role of S. oneidensis MR-1 Fur in energy metabolism that extends the traditional model of Fur as a negative regulator of iron acquisition systems.

Shewanella oneidensis MR-1 (formerly S. putrefaciens MR-1), a gram-negative facultatively anaerobic bacterium, is capable of coupling the generation of energy to the reduction of insoluble ferric iron (Fe3+), as well as other compounds (e.g., manganese, uranium, nitrate, fumarate, and dimethyl sulfoxide) in the absence of O2. Despite extensive research on electron transport-linked Fe(III) reduction, very little is known about the genetic basis and regulation of iron transport and metabolism in S. oneidensis. Sequence determination of the 5-Mb genome of S. oneidensis MR-1 has been completed recently by The Institute for Genomic Research (TIGR) with the support of the U.S. Department of Energy, thus permitting the global prediction of gene function based on sequence homology. Sequence annotation of the S. oneidensis MR-1 genome revealed a putative fur (ferric uptake regulator) gene. The annotated biological function of this gene, however, has not been verified experimentally.

In contrast to other bacteria with well-characterized fur genes, S. oneidensis MR-1 uses iron for both the biosynthesis of cellular enzymes and macromolecules (assimilatory processes) and energy production (dissimilatory processes) (46, 50). To examine the importance of Fur in regulating gene expression in a dissimilatory metal-reducing bacterium, an S. oneidensis strain (FUR1) harboring an insertional disruption in the fur gene was created and then analyzed using DNA microarrays consisting of 691 open reading frames (ORFs) and two-dimensional (2-D) gel electrophoresis in conjunction with mass spectrometry. Besides the expected derepression of iron siderophore biosynthesis and receptor genes in FUR1, the fur mutation affected the transcription of a number of genes involved in electron transport systems, energy metabolism, and regulation as well as a putative Fnr-like regulatory gene, etrA (encoding electron transport regulator A). Physiology studies, however, revealed no substantial difference between the wild type and the fur mutant in the ability to grow and utilize different electron acceptors under anaerobic conditions. While the findings reported here support previous descriptions of Fur as a negative regulator of iron acquisition genes, this study also suggests that S. oneidensis Fur plays a role in the coordinate regulation of energy metabolism.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.A list of all bacterial strains and plasmids used in this study is given in Table 1.
S. oneidensis and E. coli strains were grown in Luria-Bertani (LB) medium (59) at 30 and 37°C, respectively. When needed, the growth medium was supplemented with antibiotics at the following concentrations: for E. coli and S. oneidensis, 50 and 25 μg of kanamycin per ml, respectively; and for S. oneidensis, 10 μg of rifampin per ml. The suicide vector pKNOCK-Kmr has been described elsewhere (1).

Disruption of the fur locus in S. oneidensisThe putative fur gene was inactivated by integration of the suicide plasmid pKNOCK-Kmr (2 kb in size) into the chromosomal fur locus. An internal fragment (179 bp) of fur was amplified by PCR using the primers 5543IM-F (5"-TGCAAGGACCTGAAAACC-3") and 5543IM-R (5"-CTGAGTCGATAACTCGAATACG-3") and purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, Calif.). The amplified fragment was cloned into the SmaI site of plasmid pKNOCK-Kmr using the Perfectly Blunt cloning kit (Novagen, Madison, Wis.) according to the manufacturer's instructions, and the resulting construct, fur::pKNOCK-Kmr, was introduced into competent E. coli S17-1/λpir cells by electroporation. Transformants were screened for the correct recombinant plasmid using the Fast-Link screening kit (Epicentre Technologies, Madison, Wis.). For conjugal transfer of the suicide plasmid construct, E. coli S17-1/λpir cells harboring the fur::pKNOCK-Kmr plasmid were used in mating experiments with strain DSP10, a spontaneous rifampin-resistant derivative of S. oneidensis MR-1. E. coli transformants and DSP10 cells were grown separately in LB medium overnight, washed in fresh medium, and mixed in a 1:1 ratio by being spotted onto 0.2-μm-pore-size Millipore membrane disks. Following a 6-h incubation at room temperature, cells were removed from the filter disks, resuspended in medium, and plated onto LB agar plates supplemented with kanamycin (25 μg ml−1) and rifampin (10 μg ml−1).

Correct integration of the pKNOCK-Kmr suicide vector into the fur locus was verified by PCR amplification and reverse transcription-PCR (RT-PCR) analysis. PCR confirmation was accomplished by comparing the sizes of the products amplified from wild-type and mutant DNAs by using fur-specific primers that flanked the pKNOCK-Kmr insertion sites. The forward external primer 5543F (5"-GGTTTGAAAATCACCCTGC-3") and the reverse external primer 5543R (5"-ATTGTACTTACTGGCAATCTCG-3") were used. For RT-PCR, 2 μg of DNase I-treated total RNA from wild-type and fur mutant cells served as the template for cDNA synthesis in a reverse transcription reaction mixture containing 10 μM primer 5543R, 4 μl of 5× First Strand buffer (Gibco BRL, Gaithersburg, Md.), 1 μl of 0.1 M dithiothreitol (Gibco BRL), 10 mM deoxynucleoside triphosphates, and 200 U of Superscript II RNase H− reverse transcriptase (Gibco BRL). Reaction mixtures (total volume of 17 μl) were incubated at 37°C for 1 h. Two microliters of each reverse transcription product was used in PCR amplification with the primers 5543IM-F and 5543R.

Siderophore production and anaerobic growth on various electron acceptors.Siderophore biosynthesis and secretion by FUR1 were compared with those by the wild-type strain in LB medium with or without the addition of 50 μM FeCl3. Cultures of these strains were grown aerobically to stationary phase (optical density at 600 nm [OD600] = 4) at 30°C. Cell-free culture supernatants (500 μl) were mixed with an equal volume of chrome azurol S assay solution prepared as described previously (61) and incubated at room temperature for 2 h before the absorbance at 630 nm was measured. Siderophore production was calculated as the ratio of supernatant A630 to control (uninoculated medium) A630 from dilutions giving a linear range of absorbance.

PCR amplification and microarray constructionBecause sequence determination of the S. oneidensis MR-1 genome was not finalized at the time of this study, partial genome microarrays were constructed that contained 691 PCR-amplified MR-1 ORFs putatively involved in energy metabolism, transcriptional regulation, adaptive responses to environmental stress, iron acquisition, and transport systems (for a detailed listing, see our website at http://www.esd.ornl.gov/facilities/genomics/partial_microarrays.html). PCR primers used to amplify probable genes from S. oneidensis MR-1 were designed using the computer program Primer 3 (Whitehead Institute) with genome sequence information provided as a courtesy of TIGR (John Heidelberg, personal communication) and then synthesized at Stanford University. To simplify PCR amplifications, all primers were designed to have similar melting temperatures. Primer sequences were searched against the genome database for S. oneidensis MR-1 with FASTA to evaluate the potential for cross-amplification among different homologous genes. Amplified DNA fragments were considered correct if PCRs contained a single product of the expected size as determined by agarose gel electrophoresis. For purposes of quantification, the following control DNA samples were also included on the array: (i) a set of three serial dilutions of S. oneidensis MR-1 genomic DNA spotted onto each corner of each quadrant of the array to allow two-channel normalization of the fluorescence over a range of signal intensities, (ii) pUC19 plasmid as a negative control, (iii) non-S. oneidensis DNA from yeast as an additional negative control, (iv) internal standards derived from five yeast genes that were cloned into vectors to allow transcription from a T7 promoter, and (v) blank control spots.

PCR products (concentrations ranging from 100 to 300 ng μl−1) prepared in 50% DMSO (Sigma Chemical Co., St. Louis, Mo.) were spotted onto glass CMT-GAPS slides (Corning, Corning, N.Y.) with ChipMaker 3 pins (Telechem International, Sunnyvale, Calif.) using a PixSys 5500 robotic printer (Cartesian Technologies, Inc., Irvine, Calif.) under conditions of 62% relative humidity. PCR products representing 691 different ORFs were spotted in four replicates on a single slide. Postprocessing of the microarray slides was carried out according to the protocol of the manufacturer (Corning).

RNA isolation and preparation of fluorescein-labeled cDNA.Total cellular RNAs from wild-type and fur mutant strains of S. oneidensis grown in the presence or absence of 50 μM ferric citrate were isolated using the TRIzol Reagent (Gibco BRL) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNase I (Ambion, Inc., Austin, Tex.) to digest residual chromosomal DNA and then purified with the Qiagen RNeasy Mini kit prior to spectrophotometric quantitation at wavelengths of 260 and 280 nm.

Fluorescently labeled cDNA copies of total cellular RNA extracted from wild-type and mutant cells were prepared by incorporation of fluorescein-labeled nucleotide analogs during a first-strand reverse transcription reaction. RNA from the wild-type strain was fluorescently labeled with Cy5 (or Cy3), and that from the mutant was labeled with Cy3 (or Cy5). Two sets of duplicate reactions were carried out in which the fluorescent dyes were reversed during cDNA synthesis to minimize gene-specific dye effects. Each 30-μl labeling reaction mixture contained 10 μg of total cellular RNA; 9 μg of random hexamer primers (Gibco BRL); 10 mM (each) dATP, dGTP, and dTTP; 0.5 mM dCTP; 3 μl of 0.1 M dithiothreitol; 40 U of RNase inhibitor (Gibco BRL); 1 mM either Cy3-dCTP or Cy5-dCTP (Perkin-Elmer/NEN Life Science Products, Boston, Mass.); and 200 U of Superscript II RNase H− reverse transcriptase in 1× First Strand buffer. The reverse transcription reaction was allowed to proceed for 2 h at 42°C. The labeled cDNA probe was treated with 1 N NaOH to remove residual RNA, purified using a Qiagen PCR purification column, and concentrated in an SC110 Speedvac (Savant Instruments, Inc., Holbrook, N.Y.).

Microarray hybridization and scanning.Gene expression analysis was performed using four independent microarray experiments, with each slide containing four replicate arrays (a possible total of 16 data points per gene). The two labeled cDNA pools (wild type and mutant) to be compared were mixed and hybridized simultaneously to the array in a solution containing 3× saline sodium citrate (SSC) (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.3% sodium dodecyl sulfate (SDS), and 24 μg of unlabeled herring sperm DNA (Gibco BRL). Hybridization was carried out under a 22- by 40-mm Hybrislip (Sigma) in a waterproof CMT-slide chamber (Corning) submerged in a 65°C water bath for 12 to 15 h. Following hybridization, slides were washed with 1× SSC-0.2% SDS and 0.1× SSC-0.2% SDS for 5 min each at ambient temperature and then with 0.1× SSC for 30 s at ambient temperature prior to being air dried. Microarrays were scanned using the confocal laser microscope of the ScanArray 5000 Microarray Analysis System (GSI Lumonics, Watertown, Mass.) at a resolution of 5 μm per pixel. Scanning parameters (laser power and photomultiplier tube or PMT gain) were adjusted, so that overall intensities in both fluorescence channels were relatively equal and few spots were saturated.

Quantitative analysis of hybridization intensities and normalization.To determine fluorescence intensity (pixel density) and background intensity, 16-bit TIFF scanned images were analyzed using the software ImaGene version 3.0 (Biodiscovery, Inc., Los Angeles, Calif.). Prior to channel normalization, microarray outputs were first filtered to remove spots of poor signal quality by excluding those data points with a mean intensity of less than two standard deviations above the overall background for both channels (35). Channel normalization was accomplished using the geometric mean normalization algorithm (N. Morrison et al., Nature Genetics Microarray Meeting, Scottsdale, Ariz., 1999). Briefly, this included calculating the trimmed geometric mean (TGM) for natural log-transformed signal intensities and then calculating (ln[X] − TGM[X]) × (SDTGM X)−1, where X represents the signal intensity and SD is the standard deviation. The values were converted from log space, and fluorescence ratios (e.g., Cy5/Cy3) were determined. Log-transformed fluorescence ratios for each experiment were inspected to determine experimental quality and distribution of the ratios. The TGM normalized ratios were averaged among arrays with the fluorophores reversed (40, 73). The expression of a gene was considered significantly different if the ratio of the two fluorescent signals was greater than 2 (60).

Isoelectric focusing gels were cast as previously described by Anderson and Anderson (2), using a 2:1 mixture of pH 5 to 7 and pH 3 to 10 ampholytes (Bio-Rad). Aliquots of sample containing 20 μg of total cellular protein were loaded onto each gel. Each sample was subjected to 2-D gel electrophoresis in triplicate to control for gel-to-gel variations and to permit the application of statistical tests. Following isoelectric focusing, the gels were equilibrated in a buffer containing SDS as described previously (54). The second-dimension slab gels were cast using a linear gradient of 10 to 17% polyacrylamide. The equilibrated tube gels were secured to the slab gels using agarose, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described previously (3). The following proteins (Sigma) were added as molecular weight standards: phosphorylase b (97,400), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (30,000), trypsin inhibitor (21,000), and alpha lactalbumin (14,000). Proteins were fixed in the gels by soaking in a solution containing 50% (vol/vol) ethanol with 0.1% (vol/vol) formaldehyde and 1% (vol/vol) acetic acid for approximately 6 h and subsequently visualized by silver staining (23).

Image acquisition and analysis of 2-D gels.The 2-D gel images were digitized using an Eikonix 1412 charge-coupled device scanner interfaced with a VAX 4000 to 90 workstation. The images were then processed, and parameter lists (spot files) were generated using the Tycho II software developed at Argonne National Laboratory (4). An S. oneidensis wild-type spot file was copied to serve as the reference pattern for the experiment. On average, over 1,000 spots were detected on each 2-D gel image. Of these, approximately 800 of the most reproducible spots were included in the data analysis. All spot files (two or three two-dimensional gel patterns per cell sample) were matched to the reference pattern so that each matched spot in the patterns was numbered. Statistical analysis of the relative abundance of each matched protein spot across the data set was accomplished by using a two-tailed Student t test as described previously (24). Only those proteins showing quantitative differences with at least a probability (P) of less than 0.05 were considered to differ significantly in abundance between the wild-type and mutant samples.

Protein identification by mass spectrometry.One hundred fifty to 200 micrograms of protein was separated by isoelectric focusing. After separation in a second SDS-PAGE dimension, the proteins were detected by staining the gel with Coomassie blue R250 for 18 h. Protein spots to be identified were excised from one to five replicate gels (the number of spots required varied with the abundance of individual proteins) and processed for mass spectrometric analysis by following the procedure developed by Shevchenko et al. (62). Briefly, excised spots were reduced at room temperature with tris(2-carboxyethyl) phosphine (Pierce, Rockport, Ill.), alkylated with iodoacetamide (Sigma), and digested in situ with modified trypsin (Promega Corp., Madison, Wis.). Peptides were extracted from the gel by changes of 25 mM ammonium bicarbonate and 5% formic acid in 50% acetonitrile and then analyzed by micro-liquid chromatography-electrospray ionization tandem mass spectrometry (micro-LC-ESI-MS/MS).

For micro-LC-ESI-MS/MS, samples were loaded onto an in-house-constructed fritless 365- by 100-μm fused silica capillary column (22) packed with 5-μm Zorbax XDB-C18 packing material (Agilent Technologies, Palo Alto, Calif.) at a length of 7 to 8 cm. The flow rate at the tip was controlled to 200 to 300 nl/min using a precolumn splitter. The tryptic peptides were separated with a 30-min linear gradient of 0 to 60% solvent B (80% acetonitrile-0.02% heptafluorobutyric acid) and then entered an LCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, Calif.). Tandem mass spectra were automatically collected in the data-dependent mode during the 30-min LC-MS runs. Obtained MS/MS spectra were then directly subjected to SEQUEST (20) database searches without the need for manual interpretation. SEQUEST identified proteins in a spot by correlating experimental MS/MS spectra to protein sequence in the S. oneidensis MR-1 database (41).

Computer analyses.The sequence analysis software OMIGA 2.0 (Oxford Molecular Ltd., Oxford, England) was used to design PCR oligonucleotide primer sets for the fur gene, to assemble multiple-sequence alignments, and to search for probable ORFs. Statistical analysis of the microarray data was performed using the computer program SAS (SAS Institute, Inc., Cary, N.C.).

RESULTS

Sequence analysis of the S. oneidensis fur geneAnnotation of the genome sequence for the metal-reducing bacterium S. oneidensis MR-1 (TIGR, unpublished data) revealed the presence of a fur homolog (429 bp in size), which was predicted to be monocistronic based on an analysis of probable ORFs. Comparison of the deduced amino acid sequence showed that S. oneidensis MR-1 Fur shares a high degree of identity to its homologs in E. coli (73%) and V. cholerae (79%) and a lower degree of sequence identity to P. aeruginosa Fur (57%) (Fig. 1). This high level of homology at the primary sequence level strongly suggests that these proteins share similar biological functions. The fur gene was predicted to encode a 143-amino-acid protein with a predicted molecular mass of 16,286 Da and a pI of 5.57. A potential ribosome-binding site was located 6 nucleotides upstream of the proposed ATG translation start. Analysis of the deduced amino acid sequence of S. oneidensis MR-1 Fur also revealed the presence of two potential metal-binding domains rich in histidines and cysteines: a conserved HHHXHX2CX2C motif at amino acid residues 86 to 96 and another, less conserved carboxyl-terminal motif (HCX4CXH) at residues 132 to 140 (Fig. 1).

Transcription of the E. coli fur gene is modulated by Fur itself through a single Fur-binding site overlapping the −10 region in the promoter sequence (18). Examination of the 5" region flanking the S. oneidensis fur ORF revealed a putative Fur-binding site that overlapped a consensus −10 promoter element (TATAAT) and was positioned 40 nucleotides upstream of the presumed ATG translational start. The S. oneidensis Fur box, TATAATGGCAAGCACTATC, matched the E. coli consensus sequence (GATAATGATAATCATTATC) in 14 base positions out of 19 (a 74% identity), suggesting that transcription of S. oneidensis MR-1 fur may be controlled by an autoregulatory mechanism.

Generation of a fur mutant strain.To inactivate the fur gene, we utilized the suicide plasmid vector pKNOCK-Kmr, which contained a 179-bp internal fragment of S. oneidensis MR-1 fur. Disruption of the fur gene by suicide plasmid integration was verified by PCR amplification using fur-specific primers that flanked the pKNOCK-Kmr insertion sites. As expected, a PCR product approximately 324 bp in size was amplified from wild-type genomic DNA, and a 2.5-kb product, which was consistent with plasmid-interrupted fur, was amplified from FUR1 genomic DNA (data not shown). RT-PCR analysis demonstrated that the expected 281-bp RT-PCR product, indicative of fur expression, could be detected in wild-type cells but not in the FUR1 mutant strain (data not shown).

Phenotypic characterization of the fur mutant.Siderophore biosynthesis by the DSP10 parent and fur mutant strains of S. oneidensis was compared in LB medium with and without the addition of 50 μM FeCl3 by using the chrome azurol S assay. The fur mutant produced approximately threefold more siderophore than the wild type when grown to stationary phase under aerobic conditions. Growth of these strains in the presence of 50 μM FeCl3 reduced siderophore production by the wild-type strain to background levels (at least a 12-fold reduction), whereas siderophore production by the fur mutant remained essentially unaffected in response to increased iron levels (data not shown).

To determine whether the fur mutation affected anaerobic metabolism, the phenotype of FUR1 was also examined in terms of the ability of the strain to grow on and reduce various electron acceptors under anaerobic respiratory conditions. Based on end point culture turbidity determinations, the fur mutant resembled DSP10 in its ability to grow on the following electron acceptors: MnO2, Fe(OH)3, Fe(III) citrate, nitrate, nitrite, DMSO, TMAO, fumarate, thiosulfate, sulfite, and AQDS (data not shown). In addition, FUR1 retained the ability to reduce Fe(III) and Mn(IV) at rates that were comparable to those of DSP10 (data not shown).

Gene expression profiling of the fur mutant.Partial genome microarrays were used to identify genes in S. oneidensis MR-1 that were affected by the fur mutation. Expression arrays contained 691 different DNA elements, representing approximately 15% of the total protein-coding capacity of the MR-1 genome. These predicted ORFs had annotated functions in energy metabolism, transcriptional regulation, adaptive responses to environmental stress, iron acquisition, substrate transport systems, biosynthesis, and other cellular functions. Gene expression in the fur mutant was compared with expression in the wild-type strain under aerobic conditions in the presence of additional iron (50 μM ferric citrate). Experiments in the presence of the iron chelator 2,2"-dipyridyl (0.2 mM) were unsuccessful because of the inability of S. oneidensis MR-1 to grow sufficiently under conditions of low iron.

Following microarray data normalization and the removal of low-confidence spot data (36), a population of 331 genes was selected for further analysis. The relative transcriptional responses of each of these genes in the fur mutant were expressed as mean natural log-transformed fluorescence ratios and compared (Fig. 2). Of this subset, 30 genes consistently exhibited significant changes (>2-fold differences) in transcription relative to the wild-type control (Table 2). Coefficients of variation were calculated for each differentially expressed gene to determine the total variation in intensity ratios and the reliability of the results. Statistical analysis indicated that the expression levels were significantly different (0.01 ≤ P ≤ 0.05) from the reference for all of the genes listed in Table 2.

Analysis of microarray gene expression data. Mean signal intensity ratios of mutant to wild-type mRNA levels were obtained from two to four independent replicate experiments for each gene. Genes with mean fluorescence intensities less than two standard deviations above background in both channels were excluded. Expression ratios were transformed to natural log, such that a 2.7-fold increase (solid line) in transcript levels in the fur mutant equaled 1, whereas a 2.7-fold decrease (dashed line) equaled −1. The relative error of each expression ratio is presented.

Other genes with annotated functions in energy metabolism, transcriptional regulation, and oxidative defense also showed altered expression patterns in the fur mutant. Interestingly, the transcription of genes for a number of electron transport-associated components (ccoNQ, cytochrome c maturation protein B gene, cytochrome b561 gene, and qor) decreased 2.6- to 4.2-fold in FUR1 grown under aerobic respiratory conditions. A 2.9-fold reduction in expression was observed for etrA, a gene encoding a putative electron transport regulator that shares a high degree of primary sequence identity to E. coli Fnr (73.6%). Fnr (fumarate-nitrate reduction/regulator) activates transcription of genes encoding anaerobic respiratory functions while repressing expression from some promoters controlling transcription of aerobic respiratory enzymes (29). EtrA appears to play a role in the regulation of anaerobic respiration in S. oneidensis (47, 58), but its precise function has yet to be defined.

Analysis of protein expression profiles using 2-D PAGE and mass spectrometry.To investigate alterations in protein expression profiles as a result of the fur mutation, we compared 2-D PAGE patterns of whole-cell proteins from S. oneidensis MR-1 and FUR1 grown aerobically in LB medium (with a high concentration of iron). Representative 2-D gels of the two strains are presented in Fig. 3.
Each of the two samples was subjected to 2-D gel electrophoresis and analyzed in triplicate to enable statistical analysis. Seven major proteins (spots 362, 379, 384, 470, 681, 693, and 1099) consistently detected in both S. oneidensis strains were increased significantly (P < 0.05) in abundance in mutant cells relative to wild-type cells. A comparison of relative integrated densities averaged from two to four silver-stained gels revealed increases ranging from approximately 2.7- to 11.9-fold relative to the wild-type strain (Table 3). In addition, four protein species (spots 6, 67, 125, and 166) consistently showed decreases in abundance of FUR1 relative to the wild type (Table 3). Spots 67 and 166, in particular, displayed 4.5- and 3-fold decreases, respectively, in protein abundance.

2-D PAGE of whole-cell lysates of S. oneidensis MR-1 (A) and FUR1 (B) grown in LB medium (high concentration of iron) under aerobic conditions. Protein spots showing significant quantitative differences (a P value of at least <0.05) between the two strains are indicated by arrows and numbers. The gel images are oriented with the isoelectric focusing dimension horizontal and the SDS-PAGE dimension vertical. The approximate pI is indicated along the horizontal axis. The approximate positions of the SDS-PAGE molecular mass (MW) standards are presented in kilodaltons along the vertical axis.

Micro-LC-ESI-MS/MS was used to identify proteins showing significant differences in abundance on 2-D gels. Table 3 presents a summary of the mass spectrometry data, including predicted molecular masses, pI values, and protein identification obtained by using similarity searches based on sequence tags obtained from tryptic peptides. Three of the proteins identified (spots 125, 362, and 470) corresponded to conserved hypothetical proteins of unknown biological function, and the genes encoding these proteins were not included in the microarray experiments. The expression patterns of two other proteins, encoded by genes annotated as Fe/Mn-SOD (spot 166) and AlcC (spot 384), were consistent with their transcript levels as determined by microarray hybridization (Tables 2 and 3). The gene encoding the protein in spot 681, identified by MS/MS as a periplasmic hemin-binding protein (HutB) involved in hemin transport, was not represented on the expression array. Nonetheless, the increased abundance of the predicted HutB in FUR1 is consistent with the role of Fur as a classical transcriptional repressor of iron transport genes. Other proteins that exhibited perturbations in abundance were identified as translation elongation factor G, agglutination protein, cysteine synthase A, prismane (protein with Fe-S clusters), and a putative phosphomannomutase (Table 3). Of these proteins, only the agglutination protein-encoding gene was not represented on the microarrays. Although phosphomannomutase showed the largest increase (11.9-fold) in protein abundance, a corresponding increase in mRNA levels for the gene was not observed in the microarray analysis.

DISCUSSION

With the availability of whole-genome sequences, the next challenge is to empirically confirm the annotated functions and to provide biological meaning to genes assigned unknown functions by using integrative experimental approaches. The value of DNA expression microarrays for the analysis of genetic mutants is clear from previous studies (see, for example, references 45 and 65). In this study, microarray-based transcription profiling, physiological assays, and proteomic tools were used to investigate the effect of an insertional null mutation of the fur gene on expression patterns in the dissimilatory metal-reducing bacterium S. oneidensis MR-1.

The results described here agree with the well-established model of Fur as a negative regulator of siderophore/receptor-mediated iron acquisition and further implicate S. oneidensis MR-1 Fur in previously unidentified functions. DNA microarray analysis revealed that disruption of the Shewanella fur locus resulted in constitutive expression of genes (hxuC, alcC, fhuA, hemR, irgA, ompW, and a TonB receptor homolog gene) with annotated functions in iron transport and assimilation (Table 2). The transcription of most of these same genes (specifically, hxuC, alcC, hemR, irgA, ompW, and both fhuA genes) in wild-type S. oneidensis MR-1 was repressed during anaerobic respiratory growth in the presence of fumarate, Fe(III), or nitrate as the terminal electron acceptor, with the greatest fold reductions observed under Fe(III)-reducing conditions (8). The loss of iron repression of siderophore expression in the mutant strain, FUR1, as measured by the chrome azurol S assay, supported the microarray data and was consistent with phenotypes reported for other fur mutants (10). The two most highly derepressed genes were a predicted hxuC gene, which encodes a heme-hemopexin utilization protein C exhibiting 31% sequence identity to Haemophilus influenzae (type b) hxuC (GenBank accession no. U09840
), and alcC, which shares 48% amino acid sequence identity to its homolog (GenBank accession no. U61153
) in the Bordetella bronchiseptica alcABC operon. The H. influenzae hxuCBA operon is required for the utilization of free heme and heme bound to the human serum protein hemopexin (16, 30), whereas the alc gene cluster in Bordetella species is involved in the biosynthesis of the macrocyclic dihydroxamate siderophore alcaligin and is under the control of Fur (6, 10, 38, 39). Multiple putative Fur-binding sites were identified in the upstream regions flanking hxuC and the alc gene cluster in S. oneidensis MR-1 (Table 4). These potential Fur boxes exhibited 47 to 63% sequence identity, corresponding to matches of 9 out of 19 to 12 out of 19 to the E. coli Fur box consensus sequence. Further studies are required to determine whether these sequence elements represent functional targets for Fur-specific binding.

Genes encoding outer membrane receptor proteins (FhuA, HemR, IrgA, and TonB system receptor) also exhibited derepression in the fur mutant. The S. oneidensis irgA homolog shares 51% amino acid sequence identity to the V. cholerae virulence-associated gene irgA (26). Fur has been shown to control the expression of several virulence determinants in known microbial pathogens (13, 14), including irgA from V. cholerae (25, 71). It is important to note, however, that IrgA from V. cholerae is most closely related to iron-regulated ferric siderophore receptors (25) and therefore its likely role in virulence is in iron acquisition. Scrutiny of the promoter region for the Shewanella irgA homolog revealed two overlapping potential Fur boxes showing 47 and 68% identity to the E. coli consensus sequence (Table 4). Because of its wide distribution in nature, it is conceivable that S. oneidensis MR-1 would utilize a number of different iron transport mechanisms for its establishment in various environmental niches.

Although phenotype studies indicated that the S. oneidensis fur mutant was not impaired in growth or in the utilization of various electron acceptors under anaerobic conditions, genes involved in electron transport systems (cbb3-type cytochrome c oxidases, cytochrome c maturation protein B, cytochrome b561, and a probable quinone oxidoreductase) and the putative electron transport regulator-encoding gene, etrA, displayed decreased transcription in the FUR1 strain under aerobic respiratory conditions (Table 2). Fur box-like elements were also identified in the promoter regions for all of these genes (Table 4), with putative Fur-binding sites upstream of genes for cytochrome c maturation protein B and cytochrome b561 showing the highest sequence identity (53 to 68%) to the consensus. Interestingly, the upstream region of etrA contained an Fnr box-like sequence (TTGAT-N4-cTCgc) that displayed 70% identity to the consensus Fnr-binding site sequence (TTGAT-N4-ATCAA) (29) and overlapped a Fur box-like element with 10 matches of 19 to the consensus. The S. oneidensis etrA gene, which shows striking sequence identity to E. coli Fnr at the amino acid level, contains the four conserved cysteine residues of E. coli Fnr and the C-terminal helix-turn-helix motif that are required for iron-sulfur coordination and DNA-binding activity, respectively (58).

Despite the observation that Shewanella EtrA can complement an fnr mutant of E. coli (58), the biological role of EtrA has not been definitively resolved. Recently, Maier and Myers (47) showed that while an etrA knockout strain (ETRA-153) was able to grow on and/or reduce various electron acceptors, ETRA-153 had reduced initial growth rates on fumarate and nitrate, which correlated with lower fumarate and nitrate reductase activities. This work suggested that EtrA might play a subtle role in MR-1 anaerobic gene regulation (47). In the study described here, microarray analysis indicated that etrA transcript abundance was approximately threefold lower in aerobically grown FUR1 than in the wild type (Table 2). To determine whether the fur mutation affected initial growth rates on fumarate and nitrate, short-term anaerobic growth on these electron acceptors was examined in LB medium at 30-min intervals. In contrast to the case for the etrA mutant ETRA-153 (47), the initial growth rate of FUR1 on fumarate was comparable to that of MR-1 (Fig. 4A). The growth rates of MR-1 and FUR1 on nitrate were nearly identical over the first hour, after which the growth rate of FUR1 was approximately 62% lower than that of MR-1 (Fig. 4B). At this point, we cannot explain the difference in growth rates for FUR1 on fumarate and nitrate. Nonetheless, the microarray data and sequence analysis suggest that Fur may act with EtrA and possibly other regulatory proteins to coordinate the synthesis of iron-containing enzymes and cytochromes with iron uptake and respiration. Previous research has demonstrated that expression of E. coli sodA, the gene encoding Mn-cofactored SOD, is coordinately regulated by the global control systems of Fur, Arc, and Fnr (32). Further studies with strains harboring mutations in multiple regulatory genes are needed to confirm whether coordinate regulation of energy metabolism occurs in S. oneidensis MR-1.

Mutations in fur have been reported to have pleiotropic effects in other species (21, 43, 63, 67). Similarly, the fur mutation in S. oneidensis appeared to have a broad effect on gene expression profiles. Altered expression levels were also observed for putative transcriptional regulator genes (rpoH, phoB, and chvI), the oxidative stress protective gene sodB, and genes involved in energy/intermediary carbon metabolism (pdhC, aceE, prpC, ppc, fdhD, and yiaY), as well as other genes encoding a cation efflux system protein, 3-oxoacid coenzyme A transferase, FixG-related protein, and a NifS protein homolog (Table 2). Putative Fur box elements with weak sequence identities (37 to 58%) to the consensus were identified in the upstream regions of all of these genes (Table 4). Like that of the electron transport-associated genes, transcription of the response regulatory genes rpoH, phoB, and chvI decreased in FUR1, suggesting that expression of these genes may be under some form of Fur-mediated positive control. To our knowledge, rpoH, phoB, and chvI have not been shown to be members of the Fur regulon in other bacteria. Reduced transcript and protein expression levels for SodB (Tables 2 and 3) also suggest that positive regulation by Fur might be operative in S. oneidensis MR-1. It is important to note that E. coli Fe-SOD is positively regulated by Fur (51), and decreased Fe-SOD activity was observed in a fur mutant of P. aeruginosa (34). Finally, expression levels for prpC, ppc, fdhD, and genes encoding enzyme components of the pyruvate dehydrogenase complex (pdhC and aceE) were elevated in the S. oneidensis fur mutant, suggesting a regulatory role of Fur in the tricarboxylic acid cycle and other pathways for energy metabolism. No other report, to our knowledge, has demonstrated the effect of a fur mutation on these specific energy metabolism genes, although P. aeruginosa Fur has been shown to regulate fumarase in the tricarboxylic acid cycle (33) and a subunit of complex I (NADH:ubiquinone reductase) of the electron transport chain (53).

The effect of a fur mutation in S. oneidensis MR-1 was explored further through the use of 2-D gel electrophoresis and micro-LC-ESI-MS/MS to compare protein expression profiles in wild-type S. oneidensis and the fur mutant. Although some putative membrane-associated proteins (spots 67/ORF01553 and 125/ORF03403 in Table 3) were identified, membrane proteins were not specifically analyzed. Proteomic analysis indicated that the Fur regulon in Shewanella appears to be complex, affecting other cellular processes besides iron acquisition systems. In addition to the expected members of the Fur regulon (e.g., AlcC, HutB, and SodB), three conserved hypothetical proteins, translation elongation factor G, an agglutination protein, cysteine synthase A, prismane, and phosphomannomutase showed altered abundance levels in the fur mutant compared to the wild type. Sequence analysis indicated that the gene encoding hypothetical protein 362 clustered with genes involved in a putative sulfate transport system. Prismane is a hybrid iron-sulfur cluster protein (55, 64) and may play a role in aerobic-anaerobic respiration, although its function has not been clearly elucidated. We did not observe measurable differences in protein abundance for other genes showing differential expression on microarrays. However, it is important to note that identification of proteins was limited to those species that fell within a certain molecular mass and narrow pI range (pH 4 to 7) and were of sufficient abundance to be accurately detected and resolved in our 2-D PAGE system.

The microarray-based transcriptional profiling and proteomic analyses presented in this paper provide evidence that Fur functions as a negative regulator of siderophore production and of other genes encoding iron acquisition capabilities in S. oneidensis MR-1. Although this study is not a full description of the Fur regulon, the findings also suggest that Fur is a global regulator that appears to positively control genes involved in electron transport systems, the cellular defense against oxygen toxicity, and the regulation of certain adaptive stress responses. More research is needed to establish whether communication between Fur and other regulators such as the Fnr-like EtrA is required for the intricate coordination between intracellular iron levels and the synthesis of iron-containing proteins involved in respiration and oxygen radical detoxification.

ACKNOWLEDGMENTS

This research was supported by the U.S. Department of Energy under the Microbial Genome Program and the Natural and Accelerated Bioremediation Research Program of the Office of Biological and Environmental Research. The 2-D PAGE aspect of this work was done at Argonne National Laboratory under U.S. Department of Energy contract no. W-31-109-ENG-38. Oak Ridge National Laboratory is managed by the University of Tennessee-Battelle LLC for the Department of Energy under contract DE-AC05-00OR22725.

FOOTNOTES

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